Objective To establish a fluorescent quantitative one step real time-PCR method for the detection of GIV NoVs.Methods Specific primers and TaqMan probe of GIV NoVs were analysed and synthesized.Then the reaction system and conditions were optimized and the RNA standard was conducted according to the sequence of human GIV NoVs of our nation.The sensitivity,specificity,and repeatability of this method were evaluated,and the traditional RT-PCR method was used to assess the method.Results The sensitivity of this method reached 1.0 × 102 copies/μl,and the detection range was 102 ～ 108copies/μl.No cross reaction with NoVs (GⅠ,GⅡ),enteric adenovirus,rotavirus,astrovirus and others was found,showing high specificity.By repeated experiments,the intra-assay and inter-assay coefficient of variation (CV) about Ct value was less than 1.31％ and 3.68％ respectively,which showed good repeatability.The method has high sensitivity and accurate quantity advantage by comparison with the traditional RT-PCR on detection of positive samples.Conslusions The developed fluorescent quantitative one step real time PCR method for identification of the GIV NoVs will be applied to detecting GIV NoVs in gastroenteritis patients.

Objective To study the gene and evolution of the complete sequence of NHBGR59 strain isolated from children with diarrhea in Hebei Lulong.Methods The full-length genome was amplified based on the two contigs and reference sequence of strain of 30443 (KM198534) by methods of reverse transcription PCR and rapid-amplification of cDNA ends.The PCR products was cloned and sequenced.HBGR59 was performed on sequence analysis and phylogenetic tree.Results The complete sequence of NHBGR59 was 7563 bp (excluding PolyA tail),including three open reading frames (ORFs):ORF1 (5-5098 nt),ORF2 (5079-6731 nt) and ORF3 (6731-7510 nt),with 20 nucleotide (nt) overlapping in ORF1 and ORF2,and 1 nt overlapping in ORF2 and ORF3.By sequence analysis,NHBGR59 showed the highest homology in complete sequence with norovirus strain 30443 (98％ identity),and the ORF2 and ORF3 also showed the highest identity to strain 30443 (97.0％ and 99.0％ in nt; 98.0％ and 98.0％ in amino acid (aa)),while the ORF2 displayed the highest homology (99.0％ in nt,99.3％ in aa) with the strain 11-FJ-5 (KM461694),which was related to GⅡ.6 norovirus gastroenteritis outbreaks in Taiwan in 2014,and only one amino acid was different (aa308 D→N in P2 region) between them.The phylogenetic analysis showed NHBGR59 clustered tightly with strains of 30443 and 11-FJ-5 in norovirus genogroup Ⅱ genotype 6 (GⅡ.6).Conclusions NHBGR59 belongs to norovirus GⅡ.6,which is the mostly close to the recently reported strain 30443 and 11-FJ-5.

Objective: To analyze the prevalence, genetic structure and evolutionary characteristics of GⅡ.17 norovirus isolated from the fecal samples of rhesus monkeys in Longhu Mountain of Guangxi Zhuang Autonomous Region. Methods: A total of 400 stool specimens were collected from wild rhesus monkeys from March to August of 2015. The GⅡ.17 norovirus named as GX213 was identified in fecal samples by high-throughput sequencing technology. Reverse transcription-polymerase chain reaction was used to confirm and screen GX213, as well as amplify its complete gene sequence. Then the sequence and phylogenetic analysis of three ORFs of GX213 were constructed by software MEGA 6.0. Results: Two out of 400 fecal samples were positive. The full-length genome of GX213 was 7 565 bp (containing PloyA tail), which was composed of three open reading frames (ORFs): ORF1(10-5112 nt), ORF2(5093-6715 nt)and ORF3(6715-7494 nt), with 20 bp overlapping between ORF1 and ORF2, and 1 bp overlapping between ORF2 and ORF3.Analysis of the complete sequence of GX213 showed that it shared the highest homology with the strain of human GⅡ.17 norovirus CUHK-NS-613 (GenBank ID: KU561248) (99.5% identity), and ORF1 and ORF3 also shared the highest homology with the strain CUHK-NS-613 [99.5% and 99.4% in nucleotide (nt); 99.5% and 99.2% in amino acid (aa), respectively], which was the main cause of human norovirus outbreaks in some regions of Asia from 2014 to 2015. ORF2 sequence analysis showed that it displayed the highest identity (99.4% in nt and 99.8% in aa) to the strain CUHK-NS-491 (GenBank ID: KP698928), only one aa mutation aa₂₄₅P→S(P1.1 region) was observed in the GX213 VP1 protein. Furthermore, the phylogenetic analysis showed that GX213 was more related to CUHK-NS-613 and CUHK-NS-491 than the strain KM1509 (GenBank ID: KX356908) of GⅡ.17 norovirus recently identified in rhesus monkeys. Conclusions GX213 belongs to the human GⅡ.17 norovirus variant causing the norovirus outbreaks from 2014 to 2015. Our research suggests that GⅡ.17 norovirus can infect not only humans but also rhesus monkeys.